1. Field of the Invention
This invention relates to assays and kits for the identification of pathogenic yeasts from biological samples.
2. Description of the Prior Art
Candida albicans, other yeasts of the C. albicans clade, as well as many additional species, are of increasing importance as opportunistic pathogens in healthy as well as immunocompromised hosts. Many of these yeasts have no known sexual cycle and are endogenous organisms that can be isolated from numerous environmental sources, including the skin and mucosal tissues of persons whose immune systems are intact. However, perturbations of the immune or endocrine systems can create opportunities for Candida species to convert from a commensal state to invade tissues either locally or systemically. An example of this opportunism is the oral-esophageal or vaginal candidiasis that is encountered in physiologically stressed and in severely immunocompromised hosts such as those with HIV infections. Candida albicans is the most causative species of disseminated candidiasis followed by C. tropicalis, C. parapsilosis, and C. glabrata (Odds, Candida and Candidiasis: A Review and Bibliography, 2nd ed., Bailere Tindall, Philadelphia, 1988). Dissemination occurs when the yeast is spread via the bloodstream or by invasion of mucosal surfaces to internal organs (Odds, 1988). High-risk patient populations include individuals with malignancy or neutropenia, those receiving chemotherapy and/or multiple antibiotics, and those with indwelling catheters or low birth weight infants (Armstrong, 1989, Rev. Infect. Dis., 2:S1591-S1599).
In C. albicans, the nuclear rDNA genes encoding the 5S, 18S, 5.8S, and 28S rRNAs are found as 50-100 copy tandem repeats of approximately 10 kb unit length on chromosome seven (Magee et al., 1987, J. Bacteriol., 169:1639-1643; Thrash-Bingham and Gorman, 1992, Curr. Genetics, 22:93-100). The 5S rDNA gene (121 bp) is flanked by two nontranscribed regions located between the small and large subunits, and collectively termed the intergenic spacer (IGS). In addition, sequence analysis of the ITS1/5.8S/ITS2 internally transcribed spacer (ITS) region has shown strain variation within at least one fungal species (O'Donnell, 1992, Curr. Genet., 22:213-220), while other species have demonstrated complete conservation (Mitchell et. al., 1992, J. Med. Vet. Mycol., 30:207-218). Strain-specific restriction fragment length polymorphisms (RFLPs) have previously been observed in the IGS region for C. albicans (Magee et al., ibid).
Traditionally, many yeast infections have been detected using conventional culture techniques for isolation of the causative agent from biological samples. However, culture-based identification of Candida species requires at least one day following initial positive results to obtain a pure culture, another day to identify C. albicans isolates by germ tube formation, and two or more additional days to identify non-albicans Candida isolates by API-20C sugar assimilation strip tests and cornmeal agar morphology.
More recently, techniques have been developed for the detection of bacterial and viral DNA from the bloodstream of infected patients through the use of the polymerase chain reaction (PCR). The PCR amplifies genomic DNA geometrically so that it may be detected by agarose gel electrophoresis, Southern blotting, or dot blot hybridization (Miyakawa et al., 1992, J. Clin. Micro., 30:894-900; Kafatos et al., 1979, Nucl. Acids Res., 3:1541-1552; Lasker et al., 1992, Clin. Infect. Dis., 15:223-233).
Use of polymerase chain reaction (PCR) based tests to detect C. albicans and other pathogenic yeast DNA in body fluids has produced some encouraging results. However, routine application of these tests for the detection of candidemia remains difficult. Current methods require labor-intensive sample preparation, costly enzymes for liberation of Candida DNA, and phenol-chloroform extraction to purify DNA before PCR amplification. After amplification, detection of PCR products by gel electrophoresis or Southern blotting is often not practical in a clinical laboratory setting. Sensitivity has been variable and false positive as well as false negative results have been reported. Moreover, most studies have concentrated on the detection of C. albicans DNA but not on DNA from non-albicans Candida species.
Despite the advances in the detection of yeast infections, the need remains for a test to rapidly and accurately identify C. albicans and other pathogenic yeasts of the C. albicans clade as well as other pathogenic species.